谢瓦曲霉来源葡萄糖氧化酶的酶学性质及其比活性的优化

doi:10.19586/j.2095-2341.2025.0122

生物技术进展 ›› 2026, Vol. 16 ›› Issue (1): 123-130.DOI: 10.19586/j.2095-2341.2025.0122

谢瓦曲霉来源葡萄糖氧化酶的酶学性质及其比活性的优化

任美琪¹(), 王苑², 柏映国², 田健², 黄火清², 杨浩萌²()

^1.青岛农业大学动物科技学院，山东青岛 266109
^2.中国农业科学院北京畜牧兽医研究所，北京 100193

收稿日期:2025-09-15 接受日期:2025-11-06 出版日期:2026-01-25 发布日期:2026-02-12
通讯作者: 杨浩萌
作者简介:任美琪 E-mail: 2253778331@qq.com；
基金资助:
中央级公益性科研院所基本科研业务专项(Y2025YC53);国家肉鸡产业技术体系建设专项(CARS-41)

Enzymatic Properties of Glucose Oxidase from Aspergillus chevalieri and Optimization of its Specific Activity

Meiqi REN¹(), Yuan WANG², Yingguo BAI², Jian TIAN², Huoqing HUANG², Haomeng YANG²()

^1.College of Animal Science and Technology，Qingdao Agricultural University，Shandong Qingdao 266109，China
^2.Institute of Animal Science，Chinese Academy of Agricultural Sciences，Beijing 100193，China

Received:2025-09-15 Accepted:2025-11-06 Online:2026-01-25 Published:2026-02-12
Contact: Haomeng YANG

摘要/Abstract

摘要：

研究通过原生质体转化技术将谢瓦曲霉（Aspergillus chevalieri）来源的葡萄糖氧化酶基因（glucose oxidase，AcGox）导入黑曲霉（A. niger）菌株，获得表达的重组AcGox蛋白。结果显示，重组AcGox蛋白的最适pH为6.0，最适温度为32 ℃，表现出优良的耐酸性和较好的热稳定性。然而，AcGox的比活性仅为70.81 U·mg^-1，成为限制其应用的关键瓶颈。为解决这一问题，研究在AcGox-5表达菌株基础上，进一步过表达编码核黄素激酶和黄素腺嘌呤二核苷酸（flavin adenine dinucleotide，FAD）合成酶基因，RT-qPCR证实2个基因的转录水平显著上调。最终，优化后的菌株AcGox/FAD/FMN-3生产的Gox比活性提升至111.55 U·mg^-1，增幅达57.5%。研究证实辅因子FAD的供给水平是限制重组AcGox催化效率的关键因素，为酶工程中“辅因子工程”策略提供了直接实验证据，并为葡萄糖氧化酶的工业化生产提供了可推广的宿主改造方案，推动了Gox的应用。

关键词: 黑曲霉, 谢瓦曲霉, 葡萄糖氧化酶, 黄素腺嘌呤二核苷酸, 高效表达

Abstract:

This study introduced the AcGox gene from Aspergillus chevalieri into a Aspergillus niger strain via protoplast transformation technology， yielding the recombinant AcGox protein. Results indicated that recombinant AcGox exhibits optimal activity at pH 6.0 and 32 ℃， demonstrating excellent acid tolerance and good thermal stability. However， the specific activity of AcGox was only 70.81 U·mg^-1，to be a critical bottleneck limiting its application. To address this issue， this study further overexpressed genes encoding riboflavin kinase and FAD synthase in the AcGox-5 strain. RT-qPCR validation confirmed significantly elevated gene transcription levels. Ultimately， the specific activity of glucose oxidase produced by AcGox/FAD/FMN-3 strain increased to 111.55 U·mg^-1，with a 57.5% improvement. This study confirmed that cofactor FAD supply levels are a critical factor limiting the catalytic efficiency of recombinant AcGox. It provides direct experimental evidence for the "cofactor engineering" strategy in enzyme engineering and offers a scalable host modification approach for industrial glucose oxidase production， advancing the application of glucose oxidase.

Key words: Aspergillus niger, Aspergillus chevalieri, AcGox, flavin adenine dinucleotide, efficient expression

中图分类号:

Q936

任美琪, 王苑, 柏映国, 田健, 黄火清, 杨浩萌. 谢瓦曲霉来源葡萄糖氧化酶的酶学性质及其比活性的优化[J]. 生物技术进展, 2026, 16(1): 123-130.

Meiqi REN, Yuan WANG, Yingguo BAI, Jian TIAN, Huoqing HUANG, Haomeng YANG. Enzymatic Properties of Glucose Oxidase from Aspergillus chevalieri and Optimization of its Specific Activity[J]. Current Biotechnology, 2026, 16(1): 123-130.

图/表 8

表1 引物名称及序列信息

Table 1 Primer names and sequence information

基因名称	序列(5'→3')
VEC-F	5'-GCGGACATTCGATTTATGCCGTTATGAC-3'
VEC-R	5'-CACTACCATCGGCGCTACGG-3'
PRO-F	5'-CCGTAGCGCCGATGGTAGTGGTGGTGGGCAACTCGCTTGC-3'
PRO-R	5'-GATGGTCACGAGGGAACTTGGGCAGGGCACTGCCGAGAGAAGACAG-3'
AcGox-F	5'-GCTCTGTCTTCTCTCGGCAGTGCCCTGCCCAAGTTCCCTCGTGAC-3'
AcGox-R	5'-AGGAAGTCATAACGGCATAAATCGAATGTCCGCTTACTTGCGCGCATGATAATCCTGC-3'
FAD-F	5'-ATGACCGAGTCAATACCACCCACC-3'
FAD-R	5'-TAACGGCATAAATCGAATGTCCGCTCAGTTACGCCCCAACCGCTC-3'
FMN-F	5'-ATGCGCCCCGACCGGCCTCG-3'
FMN-R	5'-CATTTTATTTAAATTTGCGGCCGCCCATTCAGCCAGGGACAGGTTGAG-3'
PRO-R2	5'-GGTGGGTGGTATTGACTCGGTCATATCCCCAGTTGTGTATATAGAGGATTGAGG-3'
PRO-F2	5'-GTCCGAGGGCAAAGGAATAGGCCAGTTATTACCCATGAATTCATGGTGTTTTGATC-3'
PRO-R3	5'-CGCGAGGCCGGTCGGGGCGCATGGTGGAGATCCCCAGTTGTG-3'
FMN-qPCR-F	5'-TGGATTGTGAGGTCGCCAG-3'
FMN-qPCR-R	5'-ACAAAATCTCATCCCTCGCA-3'
FAD-qPCR-F	5'-GCGCTCGACAATCAAATCCA-3'
FAD-qPCR-R	5'-ACCTCAACCCCAAATGCCTA-3'
gpd-qPCR-F	5'-CCCTTCATCGAGACCCACTAT-3'
gpd-qPCR-R	5'-ACAATCAGACCCTCCTCGTAG-3'

图1 SDS-PAGE检测AcGox转化子的发酵液上清及纯化后的蛋白A：AcGox转化子的发酵液上清，M—蛋白电泳marker；泳道1~6—1~6号pPyrG-AcGox转化子的发酵液上清（4、5、6各有2瓶发酵液）；B：AcGox转化子纯化后的蛋白，泳道1—AcGox-5纯化后的浓缩蛋白；泳道2—AcGox-5浓缩蛋白稀释100倍

Fig. 1 SDS-PAGE detection of AcGox transformants fermentation broth supernatant and purified protein

图2 重组AcGox的最适温度和热稳定性A：不同温度下的酶活力；B：65~90 ℃处理后的酶活力变化

Fig. 2 The optimal temperature and thermal stability of recombinant AcGox

图3 重组AcGox的最适pH和pH稳定性A：不同pH条件下的酶活力；B：重组AcGox的pH稳定性

Fig. 3 The optimal pH and pH stability of recombinant AcGox

图4 SDS-PAGE检测转化子的发酵液上清及其比活性A：SDS-PAGE检测菌株发酵上清液；M—蛋白电泳marker；泳道CK—AcGox-5菌株发酵上清液；3、5、7、11号为AcGox/FAD/FMN菌株的发酵上清液（各有2瓶发酵液）；B：发酵液Gox的比活性；CK—AcGox-5菌株发酵液；3、5、7、11号为AcGox/FAD/FMN菌株的发酵上清液

Fig. 4 SDS-PAGE detection of transformants fermentation broth supernatant and specific activities

图5 AcGox/FAD/FMN-3菌株中黄素激酶基因和FAD合成酶基因的表达水平

Fig. 5 Expression levels of the flavin kinase gene and FAD synthase gene in AcGox/FAD/FMN-3 strain

图6 AcGox及AcGox/FAD/FMN蛋白的动力学测定A：AcGox的动力学参数；B：AcGox/FAD/FMN的动力学参数

Fig. 6 Kinetic studies of the of AcGox and AcGox/FAD/FMN

表2 动力学常数的测定

Table 2 Determination of kinetic parameters

重组Gox酶	V_max/(mol·L^-1·s^-1)	k_cat/(s^-1)	K_m/(mmol·L^-1)	k_cat/K_m/(mmol·L^-1·s^-1)
AcGox	16.77±0.81	106.90±1.44	94.24±1.28	1.13±0.12
AcGox/FAD/FMN	15.19±0.62	254.00±1.87	81.85±1.40	3.10±0.17

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谢瓦曲霉来源葡萄糖氧化酶的酶学性质及其比活性的优化

Enzymatic Properties of Glucose Oxidase from Aspergillus chevalieri and Optimization of its Specific Activity

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